Human hepatitis C virus (HCV) is a single-stranded RNA virus that causes chronic hepatitis through persistent infection. Currently, the main cause of chronic hepatitis observed worldwide is persistent HCV infection. In fact, around 50% of individuals with persistent infection develop chronic hepatitis. Chronic hepatitis in approximately 20% of these patients shifts to liver cirrhosis over the course of 10 to 20 years, and some of these patients further go on to advanced lethal pathological conditions such as hepatic cancer.
The main reasons that studies on the development of therapies for such serious diseases are hindered are the lack of efficient cellular culture systems for HCV proliferation, the lack of appropriate small animal models susceptible to HCV infection, viral replication at low levels, and genetic heterogeneity in viral genomes.
Thus, it has been expected that the development of HCV genome replication systems in cell culture systems would contribute to the understanding of viral replication and virus-cell interaction and the provision of systems for evaluation of therapeutic drugs for HCV-induced diseases.
Recently, HCV subgenomic RNA replicons have been produced as HCV-derived autonomously replicable RNAs (Patent Documents 1 and 2 and Non-Patent Documents 1-3). Thus, it becomes possible to analyze the HCV replication mechanism with the use of culture cells. Such HCV subgenomic RNA replicons are obtained by substituting structural proteins located downstream of HCV IRES in the 5′ untranslated region of HCV genomic RNA with a neomycin-resistant gene and EMCV-IRES ligated downstream thereof. By introducing such RNA replicon into a Huh7 human liver cancer cell and culturing the cell in the presence of neomycin, it was demonstrated that the RNA replicon replicates autonomously in Huh7 cells. However, only viral RNA replication, among the propagation and replication processes of HCV virus, can be evaluated in this experimental system, and thus virus particles are not produced therein. Thus, the processes of the formation of HCV virus particles in infected cells, the extracellular release of HCV particles, and infection of another cell therewith cannot be analyzed in the system.
In order to solve the above problem, a method for producing virus particles in a culture cell system has been reported. The system utilizes cDNA of HCV entire genomic RNA without the use of an RNA replicon.
Lim et al. attempted to produce HCV virus particles by treating with tetracycline a cell line obtained by introducing an expression vector in which cDNA of genomic RNA of the HCV-S1 strain (genotype 1b) is ligated downstream of a tetracycline response promoter into a Huh7 cell. They confirmed the presence of HCV particles at 1 to 6×105 copies/ml in the culture supernatant. However, they reported that such HCV particles have low infectivity (Non-Patent Document 4).
However, when HCV cDNA is expressed under the control of an RNA polymerase II-type promoter such as CMV, a CAP structure and a polyA strand are added to the 5′ end and the 3′ end of transcribed RNA, respectively. Accordingly, such RNA is used as a template for protein synthesis in a ribosome, so that replication of transcribed RNA does not take place, which is problematic.
In order to solve the above problem, Heller et al. prepared a construct that can cause intracellular synthesis of HCV RNA to which a cap and polyA are not added, by ligating a ribozyme sequence to the 5′ end and the 3′ end of the HCV genome such that it is intracellularly transcribed with RNA polymerase II and after that the transcript is cleaved with the ribozyme to produce such HCV RNA (Non-Patent Document 5). Such method for avoiding an addition of a cap at the 5′ end by means of a ribozyme is used in a method for intracellular synthesis of hairpin-type RNA (Non-Patent Document 6). In practice, it has been shown that HCV particles are produced at 1×107 copies/ml when an expression vector having an HCV construct sandwiched by two ribozymes is expressed in Huh7. Note that it has not been examined whether or not such particles exhibit infectivity.
Further, it has been recently shown that HCV particles having the ability to infect cells can be produced from HCV entire genomic RNA in a cell culture system (Patent Document 3 and Non-Patent Documents 7 and 8). In such system, the HCV particle production amount is approximately 1×107 copies/ml. Furthermore, it has been shown that it is possible to produce HCV particles having the ability to infect cells in a cell culture system with the use of chimeric viral RNA in which the non-structural protein region of the HCV con1 strain (genotype 1b) has been substituted with the gene of a viral strain (genotype 2a) (Non-Patent Document 9). No specific value for the HCV particle production amount with the use of such system has been disclosed.
Based on the above results, it has become possible to produce an experimental system that allows evaluation of the process involving the formation of HCV virus particles in infected cells, the extracellular release of HCV particles, and infection of another cell therewith.
However, the productivity of the system established by Lim et al. is low. Also, the infectivity possible with the system established by Heller et al. is unclear. Thus, it is considered that mutation might occur upon RNA replication in a system using HCV entire genomic RNA. It has been known that replication might not take place when mutation occurs in the HCV genome. In fact, it has been shown that such replication does not take place when a mutation of the GDD amino acid sequence in the NS5B protein, an HCV non-structural protein, to GND occurs. Meanwhile, the HCV particle production amount is approximately 1×107 copies/ml in both cases. Thus, further increase of the production amount has been expected.
Regarding a method for increasing HCV virus particle production amount, the production of a cell that produce a replicon at a high level has been examined. In this case, the human liver-derived Huh7 cell was used for HCV virus replication, and some cells derived from the strain were cloned. Among them, cells referred to as Huh7.5 were found to replicate approximately 3 times as many HCV RNA replicons as the parent strain (Non-Patent Document 10).
Under the above circumstances, it is thought to be important to develop a high production system for infectious HCV particles with the use of cDNA of HCV entire genomic RNA.
As a virus particle production system using cDNA corresponding to genomic RNA of an RNA virus, a system using an RNA polymerase I promoter/terminator, which is used for production of influenza virus (minus-strand RNA virus) in an animal cell system, has also been known (Non-Patent Document 11). However, it cannot be said that such influenza virus particle production system using an RNA polymerase I promoter/terminator is superior to conventional influenza virus particle production systems in terms of production amount. In addition, Non-Patent Document 11 neither describes nor suggests an HCV production system wherein HCV is a plus strand RNA virus.    Patent Document 1: JP Patent Publication (Kokai) No. 2001-17187 A    Patent Document 2: WO2004/104198A1    Patent Document 3: WO05080575A1    Non-Patent Document 1: Blight et al., Science, 290(2000) pp. 1972-74    Non-Patent Document 2: Friebe et al., J. Virol., 75(2001) pp. 12047-57    Non-Patent Document 3: Kato, T. et al., Gastroenterology, 125(2003) pp. 1808-17    Non-Patent Document 4: Lim S P. et al., Virology, 303(2002) pp. 79-99.    Non-Patent Document 5: Heller, T. et al. Proc. Natl. Acad. Sci. USA., 102 (2005) pp. 2579-83    Non-Patent Document 6: Shinagawa, T. & Ishii, S., Genes Dev., 17(2003) pp. 1340-45    Non-Patent Document 7: Wakita et al. Nature Med. 11 (2005) pp. 791-96    Non-Patent Document 8: Lindenbach B D. et al., Science. 309 (2005) pp. 623-26    Non-Patent Document 9: Pietschmann T. et al., 11th International Symposium on Hepatitis C Virus and Related Viruses, (2004)    Non-Patent Document 10: Blight, K J. et al., J. Virol., 76 (2002) pp. 13001-14    Non-Patent Document 11: Neumann, G. et al., Virology, 202 (1994) pp. 477-479